The sequence motif-specific assignment of the two distinct [2Fe-2S] clusters in rat xanthine oxidoreductase (XOR) was unequivocally established by site-directed mutagenesis of recombinant enzymes expressed in a baculovirus-insect cell system and electron paramagnetic resonance (EPR) spectroscopy. The conserved cysteine residues, including Cys-115, in the unusual C-terminal -Cys-Xaa(2)-Cys-//-Cys-Xaa(1)-Cys- motif serve as ligands to the Fe/S I center, which is probably located in close proximity to the Mo-pterin center. Other conserved cysteine residues, including Cys-43 and Cys-51, in the N-terminal plant ferredoxin-like motif serve as ligands to the Fe/S II center, which is distantly located from the Mo-pterin center. The present sequence motif-specific assignment of the Fe/S I and II centers is discussed in the light of the structural features of XOR.